When you eat and when you sleep matters: Circadian dysfunction revealed as a direct hepatic carcinogen in a humanized mouse model
Authors: Carmen Berasain
Journal: Journal of Hepatology (2023)

Circadian dysfunction induces NAFLD-related human liver cancer in a mouse modelJournal of HepatologyVol. 80Issue 2PreviewChronic circadian dysfunction increases the risk of non-alcoholic fatty liver disease (NAFLD)-related hepatocellular carcinoma (HCC), but the underlying mechanisms and direct relevance to human HCC have not been established. In this study, we aimed to determine whether chronic circadian dysregulation can drive NAFLD-related carcinogenesis from human hepatocytes and human HCC progression. Full-Text PDF See Article, pages 282–292 See Article, pages 282–292 Yes, WHEN you sleep and WHEN you eat matters, and it has a direct impact on the health of your liver. Abundant literature over the last two decades supports this tenet.1Tahara Y. Shibata S. Circadian rhythms of liver physiology and disease: experimental and clinical evidence.Nat Rev Gastroenterol Hepatol. 2016; 13: 217-226https://doi.org/10.1038/nrgastro.2016.8Crossref PubMed Scopus (168) Google Scholar, 2Mukherji A. Bailey S.M. Staels B. et al.The circadian clock and liver function in health and disease.J Hepatol. 2019; 71: 200-211https://doi.org/10.1016/j.jhep.2019.03.020Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 3Gnocchi D. Custodero C. Sabbà C. et al.Circadian rhythms: a possible new player in non-alcoholic fatty liver disease pathophysiology.J Mol Med. 2019; 97: 741-759https://doi.org/10.1007/s00109-019-01780-2Crossref PubMed Scopus (18) Google Scholar, 4Crespo M. Leiva M. Sabio G. Circadian clock and liver cancer.Cancers. 2021; 13: 3631https://doi.org/10.3390/cancers13143631Crossref PubMed Scopus (20) Google Scholar, 5Guan D. Lazar M.A. Circadian regulation of gene expression and metabolism in the liver.Semin Liver Dis. 2022; 42: 113-121https://doi.org/10.1055/a-1792-4240Crossref PubMed Scopus (7) Google Scholar, 6Daniels L.J. Kay D. Marjot T. et al.Circadian regulation of liver metabolism: experimental approaches in human, rodent, and cellular models.Am J Physiol-cell Physiol. 2023; 325: C1158-C1177https://doi.org/10.1152/ajpcell.00551.2022Crossref PubMed Scopus (2) Google Scholar, 7Bolshette N. Ibrahim H. Reinke H. et al.Circadian regulation of liver function: from molecular mechanisms to disease pathophysiology.Nat Rev Gastroenterol Hepatol. 2023; 20: 695-707https://doi.org/10.1038/s41575-023-00792-1Crossref PubMed Scopus (3) Google Scholar Indeed, indirect evidence ranging from epidemiological data to mouse models of circadian gene deficiency or induction of circadian disruption, have been provided on the role of circadian rhythm disturbances, including jet-lag, shift work and unhealthy lifestyle, in the development of chronic liver disease and hepatocellular carcinoma (HCC). Now, in this issue of the Journal of Hepatology, Padilla et al.[8]Padilla J. Osman N.M. Bissig-Choisat B. et al.Circadian dysfunction induces NAFLD-related human liver cancer in a mouse model.J Hepatol. 2024; 80: 282-292https://doi.org/10.1016/j.jhep.2023.10.018Abstract Full Text Full Text PDF Scopus (1) Google Scholar present the first experimental demonstration of the direct role of chronic circadian dysfunction as an independent carcinogen in non-alcoholic fatty liver disease (NAFLD)-related hepatocarcinogenesis in human hepatocytes. They show that a chronic human 8-hour shift work schedule induces NAFLD and NAFLD-related HCC in the humanized TIRF mouse model. Moreover, they found that this model largely recapitulates the transcriptomic reprogramming and the molecular and pathological pathways observed in patients with HCC. More importantly, they describe that the HCC transcriptome is dynamic and modifiable in vivo, and that circadian dysfunction shifts prognostic biomarkers, interfering with HCC diagnosis and treatment (Fig. 1). To adapt to the 24-hour rotation of Earth and the day and night cycle, light sensitive organisms have evolved the circadian clock system. This mechanism enables the synchronization of physiology to the environment in cycles of approximately 24 hours, where light and food are considered overarching factors regulating the expression of a network of clock genes. The central pacemaker, the suprachiasmatic nucleus located in the hypothalamus, is predominantly entrained by light signals. This master oscillator synchronizes the peripheral cell-autonomous clocks through humoral and synaptic signals. Peripheral clocks, including the liver, respond not only to those synchronizing cues emanating from the light-entrained master clock but also to rhythms in feeding and fasting.[1]Tahara Y. Shibata S. Circadian rhythms of liver physiology and disease: experimental and clinical evidence.Nat Rev Gastroenterol Hepatol. 2016; 13: 217-226https://doi.org/10.1038/nrgastro.2016.8Crossref PubMed Scopus (168) Google Scholar,[5]Guan D. Lazar M.A. Circadian regulation of gene expression and metabolism in the liver.Semin Liver Dis. 2022; 42: 113-121https://doi.org/10.1055/a-1792-4240Crossref PubMed Scopus (7) Google Scholar,[9]Pickel L. Sung H.-K. Feeding rhythms and the circadian regulation of metabolism.Front Nutr. 2020; 7: 39https://doi.org/10.3389/fnut.2020.00039Crossref PubMed Scopus (107) Google Scholar The liver is a very relevant peripheral clock influencing the whole organism as the anabolism and catabolism of nutrients and xenobiotics have a circadian rhythm. Indeed, the liver expresses the highest number of circadian genes. Around 16% of its gene transcripts are circadian[10]Zhang R. Lahens N.F. Ballance H.I. et al.A circadian gene expression atlas in mammals: implications for biology and medicine.Proc Natl Acad Sci. 2014; 111: 16219-16224https://doi.org/10.1073/pnas.1408886111Crossref PubMed Scopus (1471) Google Scholar and about 25% of all hepatic proteins show circadian oscillation in their phosphorylation status,[11]Robles M.S. Humphrey S.J. Mann M. Phosphorylation is a central mechanism for circadian control of metabolism and physiology.Cell Metab. 2017; 25: 118-127https://doi.org/10.1016/j.cmet.2016.10.004Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar highlighting the key role of transcription factors and post-translational modifications as timekeepers in hepatic cells. Recently it has been shown that the full circadian function in the liver also depends on signals emanating from other clocks.12Koronowski K.B. Kinouchi K. Welz P.-S. et al.Defining the independence of the liver circadian clock.Cell. 2019; 177: 1448-1462.e14https://doi.org/10.1016/j.cell.2019.04.025Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 13Smith J.G. Koronowski K.B. Mortimer T. et al.Liver and muscle circadian clocks cooperate to support glucose tolerance in mice.Cell Rep. 2023; 42112588https://doi.org/10.1016/j.celrep.2023.112588Abstract Full Text Full Text PDF Scopus (5) Google Scholar, 14Chen M. Lin Y. Dang Y. et al.Reprogramming of rhythmic liver metabolism by intestinal clock.J Hepatol. 2023; 79: 741-757https://doi.org/10.1016/j.jhep.2023.04.040Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar On the other hand, the liver has independent circadian functions for specific metabolic processes such as the NAD+ salvage pathway and glycogen turnover, although they still depend on the light-dark cycle.[12]Koronowski K.B. Kinouchi K. Welz P.-S. et al.Defining the independence of the liver circadian clock.Cell. 2019; 177: 1448-1462.e14https://doi.org/10.1016/j.cell.2019.04.025Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar Therefore, an entrained circadian clock is related to a healthy status. However, clock misalignments between the environment and the internal central clock, or the internal central clock and the peripheral clocks, or among the different peripheral clocks have been associated with multiple disorders. Different situations of modern life including trans-meridian travel jet-lag, shift work or high caloric eating habits, challenge our internal clocks, leading to circadian misalignments. In fact, chronic circadian disruption coupled to the epidemic of obesity could increase the risk of NAFLD-related HCC.[3]Gnocchi D. Custodero C. Sabbà C. et al.Circadian rhythms: a possible new player in non-alcoholic fatty liver disease pathophysiology.J Mol Med. 2019; 97: 741-759https://doi.org/10.1007/s00109-019-01780-2Crossref PubMed Scopus (18) Google Scholar,[6]Daniels L.J. Kay D. Marjot T. et al.Circadian regulation of liver metabolism: experimental approaches in human, rodent, and cellular models.Am J Physiol-cell Physiol. 2023; 325: C1158-C1177https://doi.org/10.1152/ajpcell.00551.2022Crossref PubMed Scopus (2) Google Scholar,[7]Bolshette N. Ibrahim H. Reinke H. et al.Circadian regulation of liver function: from molecular mechanisms to disease pathophysiology.Nat Rev Gastroenterol Hepatol. 2023; 20: 695-707https://doi.org/10.1038/s41575-023-00792-1Crossref PubMed Scopus (3) Google Scholar In this regard, properly timed feeding[7]Bolshette N. Ibrahim H. Reinke H. et al.Circadian regulation of liver function: from molecular mechanisms to disease pathophysiology.Nat Rev Gastroenterol Hepatol. 2023; 20: 695-707https://doi.org/10.1038/s41575-023-00792-1Crossref PubMed Scopus (3) Google Scholar and time-restricted feeding[9]Pickel L. Sung H.-K. Feeding rhythms and the circadian regulation of metabolism.Front Nutr. 2020; 7: 39https://doi.org/10.3389/fnut.2020.00039Crossref PubMed Scopus (107) Google Scholar have been shown to improve metabolic alterations in humans. In rodents, time-restricted feeding – with access to a high-fat diet (HFD) 8 hours per day for more than 100 days – protected mice from the adverse effects of HFD and improved oscillations of the circadian clock and the expression of their target genes, suggesting that a strict control of metabolic cycles can protect against nutritional challenge.[15]Hatori M. Vollmers C. Zarrinpar A. et al.Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet.Cell Metab. 2012; 15: 848-860https://doi.org/10.1016/j.cmet.2012.04.019Abstract Full Text Full Text PDF PubMed Scopus (1338) Google Scholar Recent data indicates that NAFLD prevalence already reaches up to 30% of the global population, and it is predicted to be the leading cause of HCC by 2030.[16]Huang D.Q. El-Serag H.B. Loomba R. Global epidemiology of NAFLD-related HCC: trends, predictions, risk factors and prevention.Nat Rev Gastroentero. 2021; 18: 223-238https://doi.org/10.1038/s41575-020-00381-6Crossref PubMed Scopus (794) Google Scholar NAFLD is a continuous spectrum of diseases, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which can progress to liver fibrosis, cirrhosis and finally to HCC. Paradoxically, 20-30% of NAFLD-related HCC occurs without cirrhosis.[17]Machado M.V. Hepatocellular carcinoma screening in NAFLD: the paradox of nearly half the cases arising in non-cirrhotic low risk patients.Ann Hepatol. 2023; 28101101https://doi.org/10.1016/j.aohep.2023.101101Crossref PubMed Scopus (2) Google Scholar The complexity of this multisystem disease makes it difficult to develop comprehensive pre-clinical animal models that fully reproduce the mechanisms of human NAFLD, and the spontaneous development of NAFLD-related HCC. This significantly impairs our understanding of the molecular mechanisms of the disease, limiting the development of prognostic biomarkers and efficient therapies. As mentioned above, accumulating evidence suggests that circadian dysfunction plays a role or accelerates the development of liver diseases such as NAFLD, cirrhosis, hepatitis and HCC, and that these disorders also disrupt clock function.[3]Gnocchi D. Custodero C. Sabbà C. et al.Circadian rhythms: a possible new player in non-alcoholic fatty liver disease pathophysiology.J Mol Med. 2019; 97: 741-759https://doi.org/10.1007/s00109-019-01780-2Crossref PubMed Scopus (18) Google Scholar,[6]Daniels L.J. Kay D. Marjot T. et al.Circadian regulation of liver metabolism: experimental approaches in human, rodent, and cellular models.Am J Physiol-cell Physiol. 2023; 325: C1158-C1177https://doi.org/10.1152/ajpcell.00551.2022Crossref PubMed Scopus (2) Google Scholar,[7]Bolshette N. Ibrahim H. Reinke H. et al.Circadian regulation of liver function: from molecular mechanisms to disease pathophysiology.Nat Rev Gastroenterol Hepatol. 2023; 20: 695-707https://doi.org/10.1038/s41575-023-00792-1Crossref PubMed Scopus (3) Google Scholar Most of this evidence comes from epidemiological data, or results from mouse models with mutant or deleted clock genes, such as Per2, Cry1/2 or BMAL1, or from mice subjected to phase-shift protocols such as restricted feeding on the light phase, or jet-lag schedules.[2]Mukherji A. Bailey S.M. Staels B. et al.The circadian clock and liver function in health and disease.J Hepatol. 2019; 71: 200-211https://doi.org/10.1016/j.jhep.2019.03.020Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar,[4]Crespo M. Leiva M. Sabio G. Circadian clock and liver cancer.Cancers. 2021; 13: 3631https://doi.org/10.3390/cancers13143631Crossref PubMed Scopus (20) Google Scholar,[6]Daniels L.J. Kay D. Marjot T. et al.Circadian regulation of liver metabolism: experimental approaches in human, rodent, and cellular models.Am J Physiol-cell Physiol. 2023; 325: C1158-C1177https://doi.org/10.1152/ajpcell.00551.2022Crossref PubMed Scopus (2) Google Scholar Along with this indirect evidence, Moore and Fu's group demonstrated in 2016 that circadian dysfunction is a direct carcinogen in mouse hepatocytes.[18]Kettner N.M. Voicu H. Finegold M.J. et al.Circadian homeostasis of liver metabolism suppresses hepatocarcinogenesis.Cancer Cell. 2016; 30: 909-924https://doi.org/10.1016/j.ccell.2016.10.007Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar Now, using the same chronic jet-lag protocol they demonstrate this direct effect in human hepatocytes.[8]Padilla J. Osman N.M. Bissig-Choisat B. et al.Circadian dysfunction induces NAFLD-related human liver cancer in a mouse model.J Hepatol. 2024; 80: 282-292https://doi.org/10.1016/j.jhep.2023.10.018Abstract Full Text Full Text PDF Scopus (1) Google Scholar Humanized livers were generated by transplanting normal human hepatocytes into male immunodeficient fumarylacetoacetate hydrolase knockout (Fah-/-) TIRF mice. TIRF mice lack Fah expression and accumulate toxic fumarylacetoacetate in hepatocytes causing liver failure and death. However, the accumulation of fumarylacetoacetate can be inhibited and mice can be maintained by administration of NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) in the drinking water. Therefore, periodic withdrawal of NTBC after human hepatocyte transplantation induces the death of mouse hepatocytes and liver regeneration from Fah+/+ human hepatocytes. Mice were kept in steady 24-hour light/dark cycles (control) or under a protocol which mimics a weekly rotating 8-hour shift work schedule (chronically jet-lagged) and were followed until 60 weeks of age. The authors found that jet-lag significantly reduced the survival of mice and the tumor latency period, and increased the incidence, growth rate and metastasis of humanized HCC (hHCC). Moreover, the pathogenesis of this jet-lag-associated hHCC was similar to human NAFLD-related HCC with glucose intolerance, abnormal hepatic fat accumulation, inflammation and fibrosis along with histologic features of NASH before HCC initiation. Importantly, these outcomes were not significantly affected by feeding jet-lag mice with a cholic acid-containing diet, reinforcing the independent carcinogenic potential of chronic circadian dysfunction. The authors provide extensive transcriptome data at different time points for humanized NASH parenchymal and non-parenchymal cells, hHCC and tumor stroma, in both control and jet-lag conditions, as well as performing comparisons with human NASH, cirrhosis and HCC transcriptomes. Interestingly, they found striking similarities with the available human data, validating the clinical relevance of this model of NAFLD-induced hepatocarcinogenesis. Moreover, they found a prognostic signature of genes differentially expressed in jet-lagged hHCC predicting HCC progression and metastasis. Circadian disruption resulted in thousands of changes in gene expression and different reprograming of human transcriptomes in NASH and HCC. Results highlighted the role of the progressive dedifferentiation of hepatocytes in hepatocarcinogenesis[19]Berasain C. Arechederra M. Argemí J. et al.Loss of liver function in chronic liver disease: an identity crisis.J Hepatol. 2022; https://doi.org/10.1016/j.jhep.2022.09.001Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar and the uncoupling of parenchymal and non-parenchymal transcriptomes. Together with metabolic reprogramming, where fatty acid and bile acid metabolic pathways were among the most deregulated gene pathways, jet-lag promoted epithelial-mesenchymal transition, inflammation and deregulation of checkpoint, oncogenic and p53 signaling. Altogether the study by Padilla et al. provides definitive evidence of the direct carcinogenic role of chronic circadian disruption in human hepatocytes. Moreover, it presents a preclinical model to assay therapeutic options and to study the molecular mechanisms of NAFLD-related hepatocarcinogenesis. Although it is a remarkable tool, there are some aspects that may be further refined. For instance, it is based on male immunodeficient mice, and it has been demonstrated that the gastrointestinal microbiome, which to an important extent depends on sex, plays a relevant role in circadian regulation.[7]Bolshette N. Ibrahim H. Reinke H. et al.Circadian regulation of liver function: from molecular mechanisms to disease pathophysiology.Nat Rev Gastroenterol Hepatol. 2023; 20: 695-707https://doi.org/10.1038/s41575-023-00792-1Crossref PubMed Scopus (3) Google Scholar Importantly, this study also highlights the relevance of taking circadian rhythms into account when evaluating prognostic markers or therapeutic strategies in patients, both as confounding factors and as points of therapeutic intervention. Grants PID2019-104265RB-I00 and PID2022-137181OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe"; CIBERehd. Please refer to the accompanying ICMJE disclosure form for further details. 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